Fig. 4

The effects of overexpression and knockdown of YAP on BMP2 induced osteoblast differentiation of MSCs. A Schematic illustration showed the experimental protocols. After knocking down YAP, BMP2 was utilized to induce osteogenic differentiation in MSCs for the purpose of studying the effects of YAP on osteoblast differentiation. Moreover, the study also evaluated whether overexpression of the constitutively activated YAP could reverse the inhibitory effect of fibroblasts on MSC osteogenic differentiation. B, C YAP and β-Actin were detected through western blot following the transfection of MSCs with adenovirus encoding shRNA of YAP (B) or YAP-5SA (C). The relative band intensity was shown below the gels and full-length gels were presented in Additional file 1: Fig. S7B, C. D After MSCs were transfected with either shRNA of YAP or scramble, 1 × 10⁴ MSCs were cultured in a single well of a 12-well plate. After the cells were treated either with a vehicle or with 200 ng/mL BMP2 for 7 days, cells were harvested and subjected to ALP staining. After the cells were treated either with a vehicle or with 200 ng/mL BMP2 for 14 days, cells were harvested and subjected to Alizarin red staining. Representative images of wells and cells were presented. Scale bars, 100 μm. E Quantitative analysis of the percentage of ALP⁺ MSCs and Alizarin red⁺ areas of D was conducted. The average percentages of ALP⁺ MSCs and Alizarin red⁺ areas were calculated from at least three fields and presented (*P<0.05; **P < 0.01). F After being transfected with adenovirus encoding YAP-5SA, MSCs were co-cultured with fibroblasts under either the BMP2 treatment or the vehicle treatment for 7 days. Subsequently, the cells were harvested and subjected to ALP staining. The representative images of the wells and cells were presented. Scale bars, 100 μm. M MSC, F fibroblast. G After being transfected with adenovirus encoding YAP-5SA, MSCs were co-cultured with fibroblasts under either the BMP2 treatment or the vehicle treatment for 14 days. Subsequently, cells were harvested and subjected to Alizarin red staining. The representative images of wells and cells were presented. Scale bars, 100 μm. M MSC, F fibroblast. H Quantitative analysis of the percentages of ALP⁺ MSCs and Alizarin red⁺ areas of F and G was conducted. The average percentages of ALP⁺ MSCs and Alizarin red⁺ areas were calculated from at least three fields and presented (*P < 0.05; **P < 0.01). I Schematic illustration showed how fibroblasts regulated the osteoblast differentiation of MSCs via modulating the nuclear-cytoplasmic shuttling of YAP. Nucleus localized YAP could favour osteoblast differentiation of MSCs under the BMP2 treatment. Fibroblasts could induce the cytoplasmic localization of YAP in MSCs to inhibit osteoblast differentiation of MSCs